Udo Bläsi

RNA chaperones, RNA annealers and RNA helicases.

RNA molecules face difficulties when folding into their native structures. In the cell, proteins can assist RNAs in reaching their functionally active states by binding and stabilizing a specific structure or, in a quite opposite way, by interacting in a non-specific manner. These proteins can either facilitate RNA-RNA interactions in a reaction termed RNA annealing, or they can resolve non-functional inhibitory structures. The latter is defined as “RNA chaperone activity” and is the main topic of this review. Here we define RNA chaperone activity in a stringent way and we review those proteins for which RNA chaperone activity has been clearly demonstrated. These proteins belong to quite diverse families such as hnRNPs, histone-like proteins, ribosomal proteins, cold shock domain proteins and viral nucleocapsid proteins. DExD/H-box containing RNA helicases are discussed as a special family of enzymes that restructure RNA or RNPs in an ATP-dependent manner. We further address the different mechanisms RNA chaperones might use to promote folding including the recently proposed theory of protein disorder as a key element in triggering RNA-protein interactions. Finally, we present a new website for proteins with RNA chaperone activity which compiles all the information on these proteins with the perspective to promote the understanding of their activity.

Reduced virulence of a hfq mutant of Pseudomonas aeruginosa O1

The Sm-like protein Hfq has been implicated in the regulation of sigmaS-dependent and sigmaS-independent genes in E. coli and in the regulation of virulence factors in both, Yersinia enterocolitica and Brucella abortus. Here, we have studied the effect of Hfq on virulence and stress response of Pseudomonas aeruginosa (PAO1). We have constructed a PAO1hfq- mutant and a PAO1hfq-rpoS- double mutant to permit distinction between direct and indirect effects of Hfq. When compared to the wild-type and the rpoS- strains, the hfq knock out strain showed a reduced growth rate and was unable to utilize glucose as a sole carbon source. Elastase activity was 80% reduced in the hfq- mutant when compared to the wild-type or the rpoS- strain, whereas alginate production seemed to be solely affected by sigmaS. The production of catalase and pyocyanin was shown to be affected in an additive manner by both, Hfq and sigmaS. Moreover, twitching and swarming mediated by typeIV pili was shown to be impaired in the hfq- mutant. When compared to PAO1 wild-type and the rpoS- mutant, the hfq- mutant decreased virulence in Galleria mellonella by a factor of 1 x 10(4) and 5 x 10(3), respectively. Likewise, when compared to wild-type, the PAO1hfq- mutant was significantly attenuated in virulence when administered intraperitoneally in mice. These results strongly suggest that Hfq is a global regulator of PAO1 virulence and stress response which is not exclusively due to its role in stimulating the synthesis of sigmaS.

Leaderless mRNAs in bacteria: surprises in ribosomal recruitment and translational control.

It is commonly believed that the translational efficiency of prokaryotic mRNAs is intrinsically determined by both primary and secondary structures of their translational initiation regions. However, for leaderless mRNAs starting with the AUG initiating codon occurring in bacteria, archaea and eukaryotes, there is no evidence for ribosomal recruitment signals downstream of the 5′‐terminal AUG that seems to be the only necessary and constant element. Studies in Escherichia coli have brought to light that the ratio of initiation factors IF2 and IF3 plays a decisive role in translation initiation of leaderless mRNA, indicating that the translational efficiency of this mRNA class can be modulated depending on the availability of components of the translational machinery. Recent data suggested that the start codon of bacterial leaderless mRNAs is recognized by a ribosome‐IF2‐fMet‐tRNA complex, an intermediate equivalent to that obligatorily formed during translation initiation in eukaryotes, which points to a conceptual similarity in all initiation pathways. In fact, the faithful translation of lead‐erless mRNAs in heterologous systems shows that the ability to translate leaderless mRNAs is an evo‐lutionarily conserved function of the translational apparatus.

Both RNase E and RNase III control the stability of sodB mRNA upon translational inhibition by the small regulatory RNA RyhB

Previous work has demonstrated that iron-dependent variations in the steady-state concentration and translatability of sodB mRNA are modulated by the small regulatory RNA RyhB, the RNA chaperone Hfq and RNase E. In agreement with the proposed role of RNase E, we found that the decay of sodB mRNA is retarded upon inactivation of RNase E in vivo, and that the enzyme cleaves within the sodB 5′-untranslated region (5′-UTR) in vitro, thereby removing the 5′ stem–loop structure that facilitates Hfq and ribosome binding. Moreover, RNase E cleavage can also occur at a cryptic site that becomes available upon sodB 5′-UTR/RyhB base pairing. We show that while playing an important role in facilitating the interaction of RyhB with sodB mRNA, Hfq is not tightly retained by the RyhB–sodB mRNA complex and can be released from it through interaction with other RNAs added in trans. Unlike turnover of sodB mRNA, RyhB decay in vivo is mainly dependent on RNase III, and its cleavage by RNase III in vitro is facilitated upon base pairing with the sodB 5′-UTR. These data are discussed in terms of a model, which accounts for the observed roles of RNase E and RNase III in sodB mRNA turnover.

Control of Fur synthesis by the non‐coding RNA RyhB and iron‐responsive decoding

The Fe2+‐dependent Fur protein serves as a negative regulator of iron uptake in bacteria. As only metallo‐Fur acts as an autogeneous repressor, Fe2+scarcity would direct fur expression when continued supply is not obviously required. We show that in Escherichia coli post‐transcriptional regulatory mechanisms ensure that Fur synthesis remains steady in iron limitation. Our studies revealed that fur translation is coupled to that of an upstream open reading frame (uof), translation of which is downregulated by the non‐coding RNA (ncRNA) RyhB. As RyhB transcription is negatively controlled by metallo‐Fur, iron depletion creates a negative feedback loop. RyhB‐mediated regulation of uof‐fur provides the first example for indirect translational regulation by a trans‐encoded ncRNA. In addition, we present evidence for an iron‐responsive decoding mechanism of the uof‐fur entity. It could serve as a backup mechanism of the RyhB circuitry, and represents the first link between iron availability and synthesis of an iron‐containing protein.

RNA chaperone activity of the Sm-like Hfq protein.

The Escherichia coli Sm‐like host factor I (Hfq) protein is thought to function in post‐transcriptional regulation by modulating the function of small regulatory RNAs. Hfq also interferes with ribosome binding on E. coli ompA messenger RNA, indicating that Hfq also interacts with mRNAs. In this study, we have used stimulation of group I intron splicing in vivo and a modified in vitro toeprinting assay to determine whether Hfq acts as an RNA chaperone. Hfq was able to rescue an RNA ‘folding trap’ in a splicing defective T4 bacteriophage td gene in vivo. Enzymatic analysis showed that Hfq affects the accessibility of the ompA start codon, as well as other bases within the ribosome‐binding site, explaining its negative effect on ribosome binding. We also show that the Hfq‐induced structural changes in ompA mRNA are maintained after proteolytic digestion of the protein, which classifies Hfq as an RNA chaperone.

Therapy of Experimental Pseudomonas Infections with a Nonreplicating Genetically Modified Phage

ABSTRACT Bacteriophage therapy of bacterial infections has received renewed attention owing to the increasing prevalence of antibiotic-resistant pathogens. A side effect of many antibiotics as well as of phage therapy with lytic phage is the release of cell wall components, e.g., endotoxins of gram-negative bacteria, which mediate the general pathological aspects of septicemia. Here we explored an alternative strategy by using genetically engineered nonreplicating, nonlytic phage to combat an experimental Pseudomonas aeruginosa infection. An export protein gene of the P. aeruginosa filamentous phage Pf3 was replaced with a restriction endonuclease gene. This rendered the Pf3 variant (Pf3R) nonreplicative and concomitantly prevented the release of the therapeutic agent from the target cell. The Pf3R phage efficiently killed a wild-type host in vitro, while endotoxin release was kept to a minimum. Treatment of P. aeruginosa infections of mice with Pf3R or with a replicating lytic phage resulted in comparable survival rates upon challenge with a minimal lethal dose of 3. However, the survival rate after phage therapy with Pf3R was significantly higher than that with the lytic phage upon challenge with a minimal lethal dose of 5. This higher survival rate correlated with a reduced inflammatory response elicited by Pf3R treatment relative to that with the lytic phage. Therefore, this study suggests that the increased survival rate of Pf3R-treated mice could result from reduced endotoxin release. Thus, the use of a nonreplicating modified phage for the delivery of genes encoding proteins toxic to bacterial pathogens may open up a new avenue in antimicrobial therapy.

Hfq‐dependent alterations of the transcriptome profile and effects on quorum sensing in Pseudomonas aeruginosa

The Pseudomonas aeruginosa quorum‐sensing (QS) systems, Las and Rhl, control the production of several virulence factors and other proteins, which are important to sustain adverse conditions. A comparative transcriptome analysis of a rpoS – and a rpoS–hfq – strain indicated that the Sm‐like RNA‐binding protein Hfq affects approximately 5% of the P. aeruginosa O1 transcripts. Among these transcripts 72 were identified to be QS regulated. Expression studies revealed that Hfq does not control the master regulators of the Las system, LasR and LasI. Upon entry into stationary phase, Hfq exerted a moderate stimulatory effect on translation of the rhlR gene and on the qscR gene, encoding a LasR/RhlR homologue. However, Hfq considerably stimulated translation of the rhlI gene, encoding the synthetase of the autoinducer N‐Butyryl‐homoserine lactone (C4‐HSL). Correspondingly, the C4‐HSL levels were reduced in a hfq– strain. To elucidate the stimulatory effect of Hfq on rhlI expression we asked whether Hfq affects the stability of the regulatory RNAs RsmY and RsmZ, which have been implicated in sequestration of the translational repressor RsmA, which in turn is known to negatively regulate RhlI synthesis. We demonstrate that Hfq binds to and stabilizes the regulatory RNA RsmY, which is further shown to bind to the regulatory protein RsmA. A model for the Hfq regulatory network is presented, wherein an alleviation of the negative effect of RsmA accounts for the observed stimulation of rhlI expression by Hfq. The model is corroborated by the observation that a rsmY– mutant mimics the hfq – phenotype with regard to rhlI expression.

Selective stimulation of translation of leaderless mRNA by initiation factor 2: evolutionary implications for translation

Translation initiation in bacteria involves a stochastic binding mechanism in which the 30S ribosomal subunit first binds either to mRNA or to initiator tRNA, fMet‐tRNAfMet. Leaderless λ cI mRNA did not form a binary complex with 30S ribosomes, which argues against the view that ribosomal recruitment signals other than a 5′‐terminal start codon are essential for translation initiation of these mRNAs. We show that, in Escherichia coli, translation initiation factor 2 (IF2) selectively stimulates translation of λ cI mRNA in vivo and in vitro. These experiments suggest that the start codon of leaderless mRNAs is recognized by a 30S–fMet‐tRNAfMet–IF2 complex, an intermediate equivalent to that obligatorily formed during translation initiation in eukaryotes. We further show that leaderless λ cI mRNA is faithfully translated in vitro in both archaebacterial and eukaryotic translation systems. This suggests that translation of leaderless mRNAs reflects a fundamental capability of the translational apparatus of all three domains of life and lends support to the hypothesis that the translation initiation pathway is universally conserved.

The small RNA PhrS stimulates synthesis of the Pseudomonas aeruginosa quinolone signal.

Quorum sensing, a cell‐to‐cell communication system based on small signal molecules, is employed by the human pathogen Pseudomonas aeruginosa to regulate virulence and biofilm development. Moreover, regulation by small trans‐encoded RNAs has become a focal issue in studies of virulence gene expression of bacterial pathogens. In this study, we have identified the small RNA PhrS as an activator of PqsR synthesis, one of the key quorum‐sensing regulators in P. aeruginosa. Genetic studies revealed a novel mode of regulation by a sRNA, whereby PhrS uses a base‐pairing mechanism to activate a short upstream open reading frame to which the pqsR gene is translationally coupled. Expression of phrS requires the oxygen‐responsive regulator ANR. Thus, PhrS is the first bacterial sRNA that provides a regulatory link between oxygen availability and quorum sensing, which may impact on oxygen‐limited growth in P. aeruginosa biofilms.